| Sustained, widespread, and highly pathogenic avian influenza (HPAI) H5N1 epidemics represent a significant public health hazard not only because they cause mortality in poultry, but also because they increase the risk of a human influenza pandemic. In the process of epidemic, millions of pourty dead, while more than 100 countries had found more than 400 human avian influenza infected cases, within which 262 patients were dead. Avain influenza have broken the species barrier and made heavy threathen to human health. Since there is no effective approaches effectively to control and prevent avian influenza, both exploring the parttern of interspecies transmission of avian influenza and seeking for effective strategy and approaches to control human avian influenza infection are of critical importance.Hemagglutinin (HA) of H5N1 binds to the receptor on the surface of host's cells, it is also the important target antigen to produce neutralizing antibodies. Finding out the neutralizing antigenic epitopes of H5N1 is the premise for the immunologic therapy of neutralizing antibodies.In present study, we firstly focus on the neutralizing antigenic epitopes of H5N1. Four HA genes isolated from avian and human cases were used to study the neutralizing epitopes. They are A/Hongkong/213/03 H5N1 HA(HK HA), A/Anhui/01/05 H5N1 HA(AH HA), A/Xinjiang/01/06 H5N1 HA(XJ HA), and A/Bar-headed Goose/Qinghai/1/05 H5N1 HA(QH HA).According to the molecular epidemiological research of HA genes, QH and XJ H5N1 strains belong to Clade2.2; AH strain belongs to Clade2.3.4; HK strain belongs to Cladel. They are the dominating epidemic Clades in China. The technical roadmap is as follows: express the HA proteins through Baculovirus expression system, develop monoclonal antibodies against QH, XJ, HK HA, and identify the neutralizing antibodies.42 hybridoma clones had been selected,7 of which were neutralizing antibodies. In this study,11 synthetic peptides containing 6 amino acids were used to identify the antibodies'binding regions through blocking assay. Results showed a majority of antibodies'binding regions focused on the front of HA. Using 20 aa peptides overlapped these regions to immunize mice and the immunized mice sera were used to do neutralization assay. The mice sera immunized by the peptides containing aa positions of 89-99 and 124-134 presented the highest titer and partial neutralizing activities. These two regions are the neutralizing epitopes of HAs.We rasied viral cross reactivity and specificity as one main line and surround three questions to study the rule of viral interspecies transmission.1. What is function of neutralizing antibody in avian influenza transmission? 2. Which one is the orignal force to drive the viral variation, antibody mediated selection pressure or receptor mediated adaptation? 3. How do the antibodies work in avian influenza interspecies transmission? There have been a number of studies comparing avian influenza on a genetic level, we collected four representative H5N1 HA genes in China, four avian influenza infected bird sera samples and ten avian influenza vaccinated or infected human sera samples. Cross reactions were investigated among HAs and MAbs or rHAs and infected sera from birds and humans. Furthermore, blockage assays of bird sera to human sera or sera to NmAbs were performed. The results showed that there was an apparent difference with regard to avian influenza in birds and humans. Since birds were the reservoir host of influenza, they demonstrated a more extensive sera recognition spectrum than humans.As a zoonotic disease, understanding the rule of avian influenza (AI) evolution in the interspecies transmission is essential for designing effective preventive measures. Two fundamental impact factors determine the direction of the viral evolution, antibodies selective pressure and receptor adaptation. A potential role of human nAbs in driving SARS-CoV evolution had been reported. Meanwhile, the receptor adaptation as a driving force also had been suggested. Several instances have demonstrated that individual amino acid mutation could induce great impact on the viral antibodies and receptor affinity in SARS-CoV. Here we utilize two natural H5N1 strains isolated from avian and human respectively with only 4 amino acid differences in hemagglutinin to investigate how avian influenza evolved into a human avian influenza under both driving forces of antibodies and receptor. A possible pathway of viral evolution in interspecies transmission has been outlined.A detailed examination of H5N1 infected human and avian sera suggests that a major driving force during interspecies transmission was neutralizing antibodies (nAbs) mediated selective pressure. Since contemporaneous and cross-strain nAb responses against H5N1 existed during natural infection, we have examined how the virus mutated under both forces of selection and adaptation by utilizing two natural H5N1 strains. These strains from human and avian respectively differ from each other at only 4 amino acids in their HAs. Mutation at aa position 90 rendered apparent changes in both the receptor specificity and antibody affinity while mutation at aa position 160 only changed the receptor specificity. Since aa position 90 was located on the side of HA tip, and aa position 160, a potential glycosylation site, was located in the receptor binding domain (RBD) on the tip of HA, we hypothesize that after the global change of HA tip by mutation at aa position 90 and then the local readjustment of RBD by mutation at aa position 160, avian influenza would be more suitable for mammal like binding pocket and make infection in human successful. In this study, we have used a strategy to detect the neutralization and binding abilities of both contemporaneous strain-specific and cross-strain nAbs against stable circulating strains and their mutants.We found that some mutations in the H5N1 hemagglutinin (HA) outside of the receptor binding domain (RBD) could induce a conformational change of the RBD to escape from nAb and change receptor preference simultaneously. These mutations were deemed as "key events" particularly during interspecies transmission to ensure the original invasion successful. They were the result of positive selection caused by antibodies. Some mutations in the RBD that only induce the change of receptor preference were deemed as "maintaining adaptation" to ensure influenza variants circulating in a new species. They were the result of adaptation caused by receptor. Our results suggest that continuing appearance of these two types of mutation made the variants persist in new specie. Examination of H5N1 infected human and avian sera had provided evidence that nAb's pressure may be a driving force for positive selection. Therefore, improved anti-viral nAb therapies focused on these positions could block the pathway of transmission in human.Taken together, our findings:Firstly, was based on a novel, effective and simple blocking peptide strategy to identify the binding regions of mAbs against H5N1 HA which were producted through baculovirus expression system;Secondly,, resulted in the first identification for two important neutralizing epitopes of H5N1 HA obtained from China, which formed a discovery patent in the process of application;Finally, revealed a pattern of interspecies transmission of avian influenza, which involves in two key amino acid positions of HA and two important mutation ways of these two positions in the process of interspecies tansmission. |
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